We propose to determine the structural requirements of the formylatable and nonformylatable E. Coli methionine (tRNAs f-tRNA Met and m-tRNA Met) for recognition by E. coli methionyl-tRNA synthetase and for attachment of methionine. In addition we propose to determine the structural requirements for recognition of Met-f-tRNA Met by E. coli methionyl-tRNA transformylase and the structural basis for binding of fMet-f-tRNA Met to the 3OS ribosomal subunit and to the 7OS ribosomal P site. The approach to be used involves chemical modification of purified tRNA, separation of the tRNA after modification into components which are active and inactive with respect to the biological activity being examined, and analysis of the structure of the active and inactive molecules for the sites of the modifications. We plan to develop methods for attachment of crosslinking reagents to a variety of sites in s-tRNA Met. These tRNAs will the be used to investigate the protein sequences in E. coli methionyl-tRNA synthetase (MRS) which are at or near tRNA binding sites and to probe the topography of ribosomal tRNA binding sites. In addition, we plan to prepare fluorescent-labeled tRNAs for studies of tRNA Met binding to MRS and to ribosomes.